Verticillium Fungicola Cell Wall Glucogactomannan-Binding of the Lectin from the Pleurotus Ostreatus Fruit Bodies

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Verticillium fungicola Cell Wall Glucogactomannan-binding of the Lectin from the Pleurotus ostreatus Fruit bodies

D. Bernardo; A. Pérez Cabo; C. García Mendoza Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

The Verticillium fungicola mycoparasitism on Agaricus bisporus fruit bodies appears to be a complex process made up of successive steps in which the recognition and binding between complementary molecules, the A. bisporus fruit body lectin and the V. fungicola cell wall glucogalactomannan, have re- cently been demonstrated. P. ostreatus fruit bodies have been described as containing a lectin and also presenting the “dry bubble” or the Verticillium disease. The aim of the present work is to purify and characterize the P. ostreatus lectin and compare the properties of both lectins in an attempt to confirm if the specific glucogalactomannan-lectin recognition and binding is the necessary step for the V. fungicola mycoparasitism process in P. ostreatus. The characteristics and properties of the purified P. o s t r e a t u s lectin together with those also previously described by us on A. bisporus lectin show that, although both lectins present different chemical struc- tures, they behave very similarly in relation to their glucogalactomannan-binding, thus confirming the existence of the specific recognition and binding step in the Verticillium disease on P. ostreatus fruit bodies.

1. Introduction

“Dry bubble” or Verticillium disease, the most serious fungal disease of the commercially grown strains of the white mushroom Agaricus bisporus, is

Genetics and Cellular Biology of Basidiomycetes-VI. A.G. Pisabarro and L. Ramírez (eds.) © 2005 Universidad Pública de Navarra, Spain. ISBN 84-9769-107-5 207 FULL LENGTH CONTRIBUTIONS caused by Verticillium fungicola. The losses in yield of A. bisporus fruit bodies in Europe produced by V. fungicola are estimated at millions of euros annual- ly. This mycopathogen infects not only A. bisporus but also other cultivated mushrooms such as Pleurotus ostreatus (Marlowe and Romaine, 1982). The only fungicide that is now used to control the disease, prochloraz, will prob- ably be banned in the near future from commercial mushroom growing be- cause V. fungicola has developed a resistance towards it (Gea et al. 1996). So, to elucidate the interaction between the mycopathogen and its host it will be necessary to know the molecular mechanisms of the infection. Bernardo et al. (2004) described that an A. bisporus fruit body purified lectin recognized and binded the isolated glucogalactomannan from cell walls of V. fungicola, suggesting the specific interaction between both organisms, prior to the secretion of V. fungicola extracellular hydrolytic enzymes conductive to the development of the disease and the further A. bisporus fruit bodies necrosis. This paper describes the characteristics and properties of a P. o s t r e a t u s fruit body lectin comparing them with those of A. bisporus fruit body lectin, in an attempt to confirm that the same molecular mechanisms of the infection occur in both mushrooms.

2. Materials and Methods

2.1. Organisms and culture conditions

Pleurotus ostreatus fruit bodies (commercial strain Amycel 3000) were grown in the CIES (Centro de Investigación, Experimentación y Servicios del Champiñón, Quintanar del Rey, Cuenca, Spain).

2.2. Purification and characterization of P. ostreatus lectin

Purification of the lectin was carried out by ammonium sulfate precipitation and ion-exchange chromatography as described previously (Bernardo et al. 2004). All procedures for characterization of the lectin (SDS-PAGE, MAL- DI-TOF mass spectrometry, chemical analysis and hemagglutination assays) have also been described before (Bernardo et al. 2004).

208 VERTICILLIUM FUNGICOLA CELL WALL GLUCOGACTOMANNAN-BINDING...

3. Results

The purification of the P. ostreatus lectin carried out following ammonium sulfate precipitation and ion-exchange chromatography is shown in Table 1. Pre- liminary experiments with ammonium sulfate fractionation showed that the hemagglutinating activity was distributed mainly in the 30%-100% saturated fraction. In the first anion-exchange chromatography all the coloured materials were absorbed by the column and the protein was eluted with the NaCl continuous gradient. In the cation-exchange chromatography the protein with the hemagglutinating activity was bound to the column, and it was eluted by means of the corresponding NaCl continuous gradient. The hemagglutinating activity evaluated at each step of purification is also shown in Table 1. SDS-PAGE analysis of the purified lectin showed that the single band obtained was a pure protein, of an apparent molecular mass of 40±4 kDa (Fig. 1). On the basis of gel filtration the native molecular mass obtained was around 80 kDa. The molecular weight of the lectin was confirmed by MAL- DI-TOF mass spectrometry, obtaining a peak of 44270 m/z (Fig 2). The sugar composition analysis showed that this protein contained a 8.15% of carbohydrate content, so it was concluded that this lectin is a dimeric glyco- protein. The sugar binding specificity of the lectin examined by hemagglutination inhibition assay is shown in Table 2. Some neutral sugars had no antagoniz- ing activity against hemagglutination, however lactose and galactose showed some effect (50 and 25 mmol L-1 respectively), and N-acetylgalactosamine and glucogalactomannan from V. fungicola cell walls treated and not treated with the fungicide prochloraz (Bernardo et al. 2002) behaved as the best in- hibitors (3.12, 6.25 and 12.5 mmol L-1 respectively).

4. Discussion

The chemical characteristics of the P. o s t r e a t u s lectin purified through this work are in good agreement with those described by Kawagishi et al. (2000) in a different strain of P. ostreatus. The specificity of this protein towards sugars has been established, and the results show that it deals with a galactose- binding lectin.

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In a previous work, Bernardo et al. (2004) showed that the A. bisporus fruit body lectin recognized and binded the glucogalactomannan from V. fungicola cell walls, suggesting that this specific interaction was essential for the further secretion of V. fungicola hydrolytic enzymes and the development of the Ver- ticillium disease on A. bisporus fruit bodies. In this report, we present evidence that the P. o s t r e a t u s fruit body lectin, although showing different chemical structure, behaves very similarly to the A. bisporus lectin in relation to its carbohydrate-binding specificity, and particularly towards the V. fungicola glucogalactomannan, thus indicating the same molecular mechanism for the V. fungicola mycoparasitism process in both A. bisporus and P. ostreatus. The strongest inhibition effect shown by the glucogalactomannan isolated from cell walls of prochloraz pretreated V. fungicola mycelium can be ex- plained by the increase of the terminal galactose residues of the molecule caused by the fungicide (Bernardo et al. 2002). Further investigations may be needed to establish if the function related to the Verticillium disease is a general role of these lectins.

5. Acknowledgements

We thank the Comision Interministerial de Ciencia y Tecnología and the Junta de Comunidades de Castilla-La Mancha for financial support, and the Centro de Investigación, Experimentación y Servicios del Champiñón (CIES) for supplying the P. ostreatus fruit bodies.

6. References

Bernardo, D., Novaes-Ledieu, M., Pérez Cabo, A., Gea Alegría, F. J. and García Men- doza, C. (2002). Effect of the fungicide Prochloraz-Mn on the cell wall structure of Verticillium fungicola. International Microbiology 5: 121-125. Bernardo, D., Pérez Cabo, A., Novaes-Ledieu, M. and García Mendoza, C. (2004). Verticillium disease or “dry bubble” of cultivated mushrooms: the Agaricus bisporus lectin recognizes and binds the Verticillium fungicola cell wall glucogalactomannan. Canadian Journal of Microbiology 50: 729-735. Gea, F. J., Tello, J. C. and Honrubia, M. (1996). In vitro sensitivity of Verticillium fungicola to selected fungicides. Mycopathologia 136: 133-137.

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Marlowe, A. and Romaine, C. P. (1982). Dry bubble of oyster mushroom caused by Verticillium fungicola. Plant Disease 66: 859-860. Kawagishi, H., Suzuki, H., Watanabe, H., Nakamura, H., Sekiguchi, T., Murata, T., Usui, T., Sugiyama, K., Suganuma, H., Inakuma, T., Ito, K., Hashimoto, Y., Ohnishi-Kameyama, M. and Nagata, T. (2000). A lectin from an edible mushroom Pleurotus ostreatus as a food intake-supressing substance. Biochimica et Biophysica Acta 1474: 299-308.

Table 1 Purification of the Pleurotus ostreatus fruit body lectin Hemagglutinating activity Fraction Protein (mg) Recovery (%) Total (units) Specific (units/mg protein)

(NH4)2SO4 335 12000 35.8 100.0 Anion-exchange 95.6 8256 86.4 68.8 Cation-exchange 12.3 4480 364.2 37.3

Table 2 Inhibition of hemagglutinating activity of Pleurotus ostreatus lectin by several carbohydrates Carbohydrate concentration (mmol/L) Carbohydrates 0.78 1.56 3.12 6.25 12.5 25 50 100 200 PBS Glucose ++++++++++ Mannose ++++++++++ Fructose ++++++++++ Lactose ++++++---+ Galactose +++++----+ Arabinose ++++++++++ Xylose ++++++++++ Rhamnose ++++++++++ N-acetyl-D-glucosamine ++++++++++ N-acetyl-D-galactosamine++------+ Glucogalactomannan* ++++-----+ Glucogalactomannan+F** + + + ------+ * glucogalactomannan of V. fungicola; ** glucogalactomannan of V. fungicola treated with the fungicide Prochloraz-Mn; +, hemagglutination positive; -, hemagglutination negative

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12 kDa

97,4

66,2

45,0

31,0

21,5

14,4 Figure 1. SDS-PAGE of the purified lectin from Pleurotus ostreatus fruit bodies: lane 1, molecular weight standards; lane 2, purified lectin.

500

400

300

200

m/z 30000 40000 50000

Figure 2. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis of the purified lectin.

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